Viral antigen nanoparticles for discriminated and quantitative detection of different subtypes of anti-virus immunoglobulins.

The aim of this study is to develop a novel method for the accurate diagnosis of the infection status of viral diseases, which requires discriminated and quantitative detection of different anti-virus immunoglubulin subtypes. Considering hepatitis A as a representative model disease, viral antigen nanoparticles (vAgNPs) were designed and synthesized by genetically presenting hepatitis A virus (HAV) antigens on the surface of human heavy chain ferritin (hFTH) nanoparticles to detect anti-HAV antibodies with discriminating immunoglobulin subtypes M and G (IgM and IgG, respectively). The vAgNPs also display multi-copies of hexa-histidine peptide (H6) on their surface to chemisorb gold ions (Au3+), which is vital for the autonomous generation of quantitatively meaningful detection signals. The quantitative level of anti-HAV IgM or IgG in 30 patient sera was successfully analyzed using the vAgNPs of HAV, which was performed through label-free one-step-immunoassay based on the self-enhancement of optical signals from gold nanoparticles clustered on the viral antigen nanoparticles. The diagnostic performance was compared with that of enzyme-linked immunosorbent assay (ELISA), which did not enable accurate quantitative assay due to the poor linearity between the antibody concentration and detection signal. Furthermore, these vAgNP-based immunoassays did not produce any false negative/positive signals, indicating 100% sensitivity and 100% specificity.

Structural basis for neutralization of hepatitis A virus informs a rational design of highly potent inhibitors.

Hepatitis A virus (HAV), an enigmatic and ancient pathogen, is a major causative agent of acute viral hepatitis worldwide. Although there are effective vaccines, antivirals against HAV infection are still required, especially during fulminant hepatitis outbreaks. A more in-depth understanding of the antigenic characteristics of HAV and the mechanisms of neutralization could aid in the development of rationally designed antiviral drugs targeting HAV. In this paper, 4 new antibodies-F4, F6, F7, and F9-are reported that potently neutralize HAV at 50% neutralizing concentration values (neut50) ranging from 0.1 nM to 0.85 nM. High-resolution cryo-electron microscopy (cryo-EM) structures of HAV bound to F4, F6, F7, and F9, together with results of our previous studies on R10 fragment of antigen binding (Fab)-HAV complex, shed light on the locations and nature of the epitopes recognized by the 5 neutralizing monoclonal antibodies (NAbs). All the epitopes locate within the same patch and are highly conserved. The key structure-activity correlates based on the antigenic sites have been established. Based on the structural data of the single conserved antigenic site and key structure-activity correlates, one promising drug candidate named golvatinib was identified by in silico docking studies. Cell-based antiviral assays confirmed that golvatinib is capable of blocking HAV infection effectively with a 50% inhibitory concentration (IC50) of approximately 1 µM. These results suggest that the single conserved antigenic site from complete HAV capsid is a good antiviral target and that golvatinib could function as a lead compound for anti-HAV drug development.

Evidence for positive selection of hepatitis A virus antigenic variants in vaccinated men-having-sex-with men patients: Implications for immunization policies.

BACKGROUND: A huge outbreak in the men-having-sex-with-men (MSM) has hit Europe during the years 2016-2018. Outbreak control has been hampered by vaccine shortages in many countries, and to minimize their impact, reduction of antigen doses has been implemented. However, these measures may have consequences on the evolution of hepatitis A virus (HAV), leading to the emergence of antigenic variants. Cases in vaccinated MSM patients have been detected in Barcelona, opening the possibility to study HAV evolution under immune pressure. METHODS: We performed deep-sequencing analysis of ten overlapping fragments covering the complete capsid coding region of HAV. A total of 14578255 reads were obtained and used for the analysis of virus evolution in vaccinated versus non-vaccinated patients. We estimated maximum and minimum mutation frequencies, and Shannon entropy in the quasispecies of each patient. Non-synonymous (NSyn) mutations affecting residues exposed in the capsid surface were located, with respect to epitopes, using the recently described crystal structure of HAV, as an indication of its potential role in escaping to the effect of vaccines. FINDINGS: HAV evolution at the quasispecies level, in non-vaccinated and vaccinated patients, revealed higher diversity in epitope-coding regions of the vaccinated group. Although amino acid replacements occurring in and around the epitopes were observed in both groups, their abundance was significantly higher in the quasispecies of vaccinated patients, indicating ongoing processes of fixation. INTERPRETATION: Our data suggest positive selection of antigenic variants in some vaccinated patients, raising concerns for new vaccination polices directed to the MSM group.

Establishment of detection antibodies BRRs batch 4 for in vitro potency assay of hepatitis A vaccines by ELISA.

The European Pharmacopoeia (Ph. Eur.) standard ELISA method for determination of antigen content of hepatitis A vaccines (HAV) requires specific coating and detection Biological Reference Reagents (BRRs). The 3rd batch of detection antibodies BRRs was established in 2015 for use in conjunction with the Ph. Eur. general chapter 2.7.14 'Assay of hepatitis A vaccine'. Stocks of these BRRs were running low and therefore the European Directorate for the Quality of Medicines & HealthCare (EDQM) organised a collaborative study to qualify replacement batches. The candidate BRR antibodies batch 4 were prepared under appropriate conditions from starting materials similar to previous batches to ensure continuity. During the collaborative study, the new batches of antibodies were compared to previous batches of BRRs. Results confirmed that they were suitable to be used for the intended purpose, and could be used at the same final concentrations as the previous batch, i.e. 1:500 for the primary antibody and 1:400 for the conjugated secondary antibody. They were adopted in June 2017 by the Ph. Eur. Commission as Hepatitis A virus primary detection antibody BRR batch 4 and Conjugated secondary detection antibody BRR batch 4, respectively. They are available from the EDQM as Hepatitis A vaccine ELISA detection antibodies set BRR batch 4.

Establishing the 1st Chinese National Standard for inactivated hepatitis A vaccine.

A reference standard calibrated in the International Units is needed for the quality control of hepatitis A vaccine. Thus, National Institutes for Food and Drug Control launched a project to establish a non-adsorbed inactivated hepatitis A vaccine reference as the working standard calibrated against the 1st International Standard (IS). Two national standard candidates (NSCs) were obtained from two manufacturers, and designated as NSC A (lyophilized form) and NSC B (liquid form). Six laboratories participated in the collaborative study and were asked to use their in-house validated enzyme-linked immunosorbent assay methods to detect hepatitis A vaccine antigen content. Although both exhibited good parallelism and linear relationship with IS, NSC B showed a better agreement among laboratories than NSC A. And based on suitability of the candidates, NSC B was selected. The accelerated degradation study showed that NSC B was stable at the storage temperature (≤-70 °C). Therefore NSC B was approved as the first Chinese national antigen standard for inactivated hepatitis A vaccine, with an assigned antigen content of 70 IU/ml.

Epitope-based recombinant diagnostic antigen to distinguish natural infection from vaccination with hepatitis A virus vaccines.

Hepatitis A virus (HAV) infection can stimulate the production of antibodies to structural and non-structural proteins of the virus. However, vaccination with an inactivated or attenuated HAV vaccine produces antibodies mainly against structural proteins, whereas no or very limited antibodies are produced against the non-structural proteins. Current diagnostic assays to determine exposure to HAV, such as the Abbott HAV AB test, detect antibodies only to the structural proteins and so are not able to distinguish a natural infection from vaccination with an inactivated or attenuated virus. Here, we constructed a recombinant tandem multi-epitope diagnostic antigen (designated 'H1') based on the immune-dominant epitopes of the non-structural proteins of HAV to distinguish the two situations. H1 protein expressed in Escherichia coli and purified by affinity and anion exchange chromatography was applied in a double-antigen sandwich ELISA for the detection of anti-non-structural HAV proteins, which was confirmed to distinguish a natural infection from vaccination with an inactivated or attenuated HAV vaccine.

Using immunoglobulin Y as an alternative antibody for the detection of hepatitis A virus in frozen liver sections

An increasing amount of research has been conducted on immunoglobulin Y (IgY) because the use of IgY offers several advantages with respect to diagnostic testing, including its easy accessibility, low cost and translatability to large-scale production, in addition to the fact that it can be ethically produced. In a previous work, immunoglobulin was produced and purified from egg yolks (IgY) reactive to hepatitis A virus (HAV) antigens. In the present work, this anti-HAV-specific IgY was used in an indirect immunofluorescence assay to detect viral antigens in liver biopsies that were obtained from experimentally infected cynomolgus monkeys. Fields that were positive for HAV antigen were detected in liver sections using confocal microscopy. In conclusion, egg yolks from immunised hens may be a reliable source for antibody production, which can be employed for immunological studies.

Using immunoglobulin Y as an alternative antibody for the detection of hepatitis A virus in frozen liver sections.

An increasing amount of research has been conducted on immunoglobulin Y (IgY) because the use of IgY offers several advantages with respect to diagnostic testing, including its easy accessibility, low cost and translatability to large-scale production, in addition to the fact that it can be ethically produced. In a previous work, immunoglobulin was produced and purified from egg yolks (IgY) reactive to hepatitis A virus (HAV) antigens. In the present work, this anti-HAV-specific IgY was used in an indirect immunofluorescence assay to detect viral antigens in liver biopsies that were obtained from experimentally infected cynomolgus monkeys. Fields that were positive for HAV antigen were detected in liver sections using confocal microscopy. In conclusion, egg yolks from immunised hens may be a reliable source for antibody production, which can be employed for immunological studies.

Establishment of hepatitis A detection antibodies set BRR batch 3 for antigen content determination by ELISA.

The current batch of the European Pharmacopoeia (Ph. Eur.) Biological Reference Reagents (BRRs) used for the in vitro potency assay of hepatitis A vaccines (HAV) by ELISA (enzymelinked immunosorbent assay) was established in 2012 for use in conjunction with Ph. Eur. general chapter 2.7.14 Assay of hepatitis A vaccine. It is composed of a coating reagent and a set of detection antibodies. As stocks of the latter are running low, the European Directorate for the Quality of Medicines & HealthCare (EDQM) organised a collaborative study to qualify replacement batches. The candidate BRR antibodies (primary monoclonal antibody and labelled secondary antibody) were prepared under appropriate conditions from starting materials similar to those used for the current batches. The new batches of antibodies were tested alongside previous batches of BRRs to ensure continuity, and the results confirmed that they were suitable for use in the potency assay of hepatitis A vaccines by ELISA using the standard method referenced in Ph. Eur. general chapter 2.7.14 at the same final concentrations as the previous batches, i.e. 1:500 for the primary monoclonal antibody and 1:400 for the secondary conjugated antibody. The outcome of the study allowed their establishment by the Ph. Eur. Commission in March 2015 as anti-hepatitis A virus primary detection antibody BRR batch 3 and conjugated secondary detection antibody BRR batch 3 respectively. They are available from the EDQM as hepatitis A vaccine ELISA detection antibodies set BRR batch 3.

[The hepatitis A virus: structural and functional organization of the genome, its molecular diagnostic value and cultivation].

The analysis of recently published data on hepatitis A virus (HAV) genome clinical features, molecular diagnostic value and cell culture propagation are reviewed. The growing need in the study of the genetic diversity of HAV isolates and the search of its possible new antigenic variants are underlined. The results of the cultivation of different HAV strains are analyzed for possible application in vaccine and diagnostic kit production.

Development of recombinant single-chain variable fragment against hepatitis A virus and its use in quantification of hepatitis A antigen.

Phage display technology has been utilized for identification of specific binding molecules to an antigenic target thereby enabling the rapid generation and selection of high affinity, fully human antibodies directed towards disease target appropriate for antibody therapy. In the present study, single chain Fv antibody fragment (scFv) to hepatitis A virus (HAV) was selected from phage displayed antibody library constructed from peripheral blood lymphocytes (PBLs) of a vaccinated donor. The variable heavy (V(H)) and light chains (V(L)) were amplified using cDNA as template, assembled into scFv using splicing by overlap extension PCR (SOE PCR) and cloned into phagemid vector as a fusion for display of scFv on bacteriophage. The phage displaying antibody fragments were subjected to three rounds of panning with HAV antigen on solid phase. High affinity antibodies reactive to hepatitis A virus were identified by phage ELISA and cloned into a bacterial expression vector pET20b. The scFv was purified by immobilized metal affinity chromatography (IMAC) on a nickel-nitrilotriacetic acid (NTA) agarose column and characterized. The binding activity and specificity of the scFv was established by its non-reactivity towards other human viral antigens as determined by ELISA and immunoblot analysis. The scFv was further used in the development of an in-house IC-ELISA format in combination with a commercially available mouse monoclonal antibody for the quantification of hepatitis A virus antigen in human vaccine preparations. The adjusted r² values obtained by subjecting the values obtained by quantification of the NIBSC standards using the commercial and the in-house ELISA kits by regression analysis were 0.99 and 0.95. 39 vaccine samples were subjected to quantification using both the kits. Regressional statistical analysis through the origin of the samples indicated International Unit (IU) values of 0.0416x and 0.0419x, respectively for the commercial and in-house kit respectively.

MEMS biosensor for detection of Hepatitis A and C viruses in serum.

Resonant microcantilever arrays are developed for the purpose of label-free and real-time analyte monitoring and biomolecule detection. MEMS cantilevers made of electroplated nickel are functionalized with Hepatitis antibodies. Hepatitis A and C antigens at different concentrations are introduced in undiluted bovine serum. All preparation and measurement steps are carried out in the liquid within a specifically designed flowcell without ever drying the cantilevers throughout the experiment. Both actuation and sensing are done remotely and therefore the MEMS cantilevers have no electrical connections, allowing for easily disposable sensor chips. Actuation is achieved using an electromagnet and the interferometric optical sensing is achieved using laser illumination and embedded diffraction gratings at the tip of each cantilever. Resonant frequency of the cantilevers in dynamic motion is monitored using a self-sustaining closed-loop control circuit and a frequency counter. Specificity is demonstrated by detecting both Hepatitis A and Hepatitis C antigens and their negative controls. This is the first report of Hepatitis antigen detection by resonant cantilevers exposed to undiluted serum. A dynamic range in excess of 1000 and with a minimum detectable concentration limit of 0.1ng/ml (1.66pM) is achieved for both Hepatitis A and C. This result is comparable to labeled detection methods such as ELISA.

Changing epidemiology of hepatitis A infection in Izmir, Turkey.

The seroprevalence study was conducted in order to determine the current seroepidemiology hepatitis A in Izmir, Turkey and to evaluate the epidemiological shift in HAV serostatus. Blood samples collected from 595 subjects aged 1-60 years were analyzed for anti-HAV IgG antibodies. The current study results were compared with those of a previous study conducted in 1998 involving the same location. There was a marked decrease in the prevalence of anti-HAV between 1998 and 2008. While anti-HAV seroprevalence rates in the current study were 4.6% in children aged 1-4 years, 23% in children aged 10-14 years, and 85% in young adults aged 20-29 years, the prevalence rates were 36% in the 1-4 years age group, 65% in the 10-14 years age group, and 95% in young adults in the previous study, indicating a shift in HAV seroprevalence from the younger to the higher age groups. As HAV infection in childhood is decreasing, the pool of susceptible adolescents and young adults is increasing in Izmir, Turkey. The majority of adolescent population is susceptible to HAV infection. The potential risk of HAV epidemics still exists. The situation of Turkey, suggested to need for mass immunization. Also, introduction of hepatitis A vaccination into the national immunization schedule of Turkey should be considered.

[The characterization analysis of HAV recombinant antigen was expressed by vaccinia virus vector].

OBJECTIVE: To find a suitable cell line for hepatitis A antigen expressed by vaccinia virus vector and to find a way of inactivation and preservation of the HAV recombinant antigen. Methods Series of cell lines such as K4,143, HEL, Hep-2 and Vero were inoculated with vaccinia virus that can express HAV recombinant antigen. ELISA was used to determine the contents of expression antigen. The characterization of the HAV antigen expressed by vaccinia virus was then analyzed after it was treated with different methods. RESULTS: The expression of HAV recombinant antigen in K4,143 and HEL cell lines was a little more than expression in Hep-2 and Vero cell lines. The antigenicity is obviously higher when HAV recombinant antigen was inactivated by beta-propiolactone other than it was inactivated by formalin. It was best to preserve the prepared HAV recombinant antigen under -40 degrees C condition. CONCLUSIONS: The application of vaccinia virus vector in hepatitis A antigen preparation was very useful and promising.

Experimental hepatitis A virus (HAV) infection in cynomolgus monkeys (Macaca fascicularis): evidence of active extrahepatic site of HAV replication.

This work studied the replication sites of hepatitis A virus (HAV) in cynomolgus monkeys (Macaca fascicularis) after intravenous inoculation. The cynomolgus monkeys were inoculated with the Brazilian hepatitis A virus strain (HAF-203). Monkeys were euthanized on days 15, 30, 45 and 60 postinoculation (pi). Liver samples, submandibular salivary gland, mesenteric lymph node and tonsils were removed for virological and pathological evaluation. Immunofluorescence analyses on liver and salivary gland sections using confocal laser scanning microscopy revealed the presence of HAV antigen (HAV Ag). The presence of HAV genome was monitored by real-time PCR. The HAV RNA was detected at 7 days postinoculation (dpi), concomitantly in serum, saliva and faeces. The highest HAV viral load was observed in faeces at 15 dpi (10(5) copies/ml), followed by serum viral load of 10(4) copies/ml at 20 dpi and saliva viral load of 10(3 )copies/ml at 7 dpi. The animals showed first histological and biochemical signs of hepatitis at 15 dpi. The HAV antigen (Ag) was present from day 7 until day 60 pi in the liver and salivary glands. The HAV replicative intermediate was also detected in the liver (4.5 x 10(4) copies/mg), salivary glands (1.9 x 10(3) copies/mg), tonsils (4.2 x 10(1) copies/mg) and lymph nodes (3.4 x 10(1) copies/mg). Our data demonstrated that the salivary gland as an extrahepatic site of early HAV replication could create a potential risk of saliva transmitted infection. In addition, the cynomolgus monkey was confirmed as a suitable model to study the pathogenesis of HAV human infection.

[Features of antigen-antibody interaction during use of linear synthetic peptides and multipeptide antigen modeling antigenic determinants of hepatitis A virus].

AIM: To study features of antigen-antibody interaction during use of linear synthetic peptides and multipeptide antigen modelling antigenic determinants of hepatitis A virus (HAV) and to evaluate perspectives for use of heterogeneous tetrameric multipeptide antigens for detection of HAV serological markers. MATERIALS AND METHODS: Linear peptides VP1 and VP3 were synthesized by fluorenylmethyloxycarbonyl (Fmoc)-polyamide solid phase method. MAP4 (VP1+VP3) was synthesized according to 9-Fmoc strategy. Interaction of these peptides with anti-HAV IgM positive sera from patients with HA was studied by noncompetitive and competitive methods of immunoenzyme assay. RESULTS. Using immunoenzyme assay, high heterogeneity of immune response in patients with HA (62 and 67% in two groups) was shown. MAP4 (VP1+VP3), unlike the combination of linear peptides VP1 and VP3, interacted with anti-HAV IgM in 41 - 45% of sera and, at the same time, did not lead to false positive results. CONCLUSION: Population of HAV is not so uniform which is usually assumed. It could be reasonable to use heterogenous multipeptide antigens, including those containing VP1 (11 - 25 a.r.) and VP3 (110 - 121 a.r.), for the development of new assays for HA diagnostics.

The role of injection versus socioeconomic factors in hepatitis A virus infection among young heroin users: Implications for vaccination policies.

The aim of this study was to determine whether heroin users have a higher prevalence of HAV infection than the general population in Spain, and whether injection is an independent risk factor. A cross-sectional cohort study was conducted between April 2001 and December 2003 in Spain that included 953 current heroin users aged 18-30 years. Dried blood spot samples were tested for HAV by ELISA. The prevalence of HAV infection (35.5%) was higher than in the general population of the same age. The logistic regression analysis did not show association between HAV infection and injection. HAV infection was associated with low educational level (OR=4.8; 95% CI=2.1-10.9) and other low-income variables. Injection is not an independent risk factor for HAV infection; rather, the principal determinants are socioeconomic factors. Consequently, HAV vaccination should be recommended not only in IDUs but also in non-IDUs depending on their socioeconomic characteristics.

Hepatitis A, B, and C infection in a community of sub-Saharan immigrants living in Verona (Italy).

BACKGROUND: In Italy, about 5% of the population is represented by immigrants. The epidemiology of hepatitis A virus (HAV), hepatitis B virus (HBV), and hepatitis C virus (HCV) infection in Africa is very different from Europe; the present study aimed to assess the seroprevalence of viral hepatitis infections in sub-Saharan African immigrants living in Verona. METHODS: A total of 182 illegal immigrants were interviewed concerning sociodemographic characteristics and epidemiological information. Their serum was tested for anti-HAV [immunoglobulin (Ig) G and IgM], HBV (HBsAg, anti-HBs, anti-HBc, HBeAg, and anti-HBe), and HCV (anti-HCV) markers. RESULTS: The immigrants (age: 3 mo-60 y) were mostly single and males, with a higher education; only 50% of them declared having a regular job. Anti-IgG HAV+ prevalence was 99.5% (100% HAV positivity in the younger age bracket). As for HBV, 67.6% (123) of the immigrants were naturally infected and 9.3% had chronic infection; 4.4% were anti-HBs+ isolated (vaccinated). For HBV infection (any HBV marker), a significant difference was only found for increasing age ( p < 0.01) and married people ( p < 0.001). A statistically significant prevalence of HBsAg was found among the unemployed ( p < 0.001) and those with a lower education ( p < 0.05). Five cases (2.7%) resulted in HCV+ with no reported specific risk factors and with no significantly different sociodemographic features; these people tended to report a low level of education and unemployment. CONCLUSIONS: HAV and HBV positivity is higher than in the autochthonous population. While HAV positivity merely represents past infection, the high prevalence of HBsAg in immigrants and the presence of HBsAg/HBeAg in the same group may represent a risk for HBV transmission. The HCV positivity rate resulted similar to the prevalence of the Italian population.